Discovery of seven novel putative antigens in membranous nephropathy and membranous lupus nephritis identified by mass spectrometry
Authors: Tiffany N. Caza, Aaron J. Storey, Samar I. Hassen, Christian Herzog, Rick D. Edmondson, John M. Arthur, Daniel J. Kenan, Christopher P. Larsen
Journal: Kidney International (2023)

Multiple autoantigens have been identified in membranous nephropathy (MN) by tissue-based proteomics. However, antigenic targets of disease are unknown for over 10% of patients with MN and over half of those with membranous lupus nephritis (MLN). Here, we identified multiple new targets in PLA2R-/THSD7A-/EXT-/NELL1-quadruple negative MN biopsies through mass spectrometry of immune complexes recovered from biopsy tissue of patients with MN. Patients with MN negative for these four antigens were identified from Arkana Laboratories case archives. Protein G immunoprecipitation recovered immune complexes from frozen biopsy tissue from 142 quadruple-negative cases and 278 cases of known antigen type, followed by interrogation by mass spectrometry. Potential putative antigens were confirmed through paraffin immunofluorescence and co-localization with IgG within immune deposits. Consecutive series of 165 cases of PLA2R-negative MN and 142 MLN biopsies were screened to determine the frequency for each potential antigen. Seven putative antigens were discovered within immune complexes from biopsies of patients with MN including FCN3, CD206, EEA1, SEZ6L2, NPR3, MST1, and VASN. Peptides from these proteins were not enriched in the 278 cases of known antigen type. Between three to 30 unique peptides were detected for each new target. Frequencies of each biomarker, determined by staining consecutive case series, ranged from under 1 to 4.9%. NPR3 and CD206 were only positive in index cases. All cases showed co-localization of IgG within the immune deposits. Thus, seven putative antigens were newly identified in MN and MLN. Due to the number of antigens identified, it is becoming impractical to type PLA2R-negative MN or MLN cases through immunostaining alone. A multiplex approach is needed for subtyping of these diseases. Multiple autoantigens have been identified in membranous nephropathy (MN) by tissue-based proteomics. However, antigenic targets of disease are unknown for over 10% of patients with MN and over half of those with membranous lupus nephritis (MLN). Here, we identified multiple new targets in PLA2R-/THSD7A-/EXT-/NELL1-quadruple negative MN biopsies through mass spectrometry of immune complexes recovered from biopsy tissue of patients with MN. Patients with MN negative for these four antigens were identified from Arkana Laboratories case archives. Protein G immunoprecipitation recovered immune complexes from frozen biopsy tissue from 142 quadruple-negative cases and 278 cases of known antigen type, followed by interrogation by mass spectrometry. Potential putative antigens were confirmed through paraffin immunofluorescence and co-localization with IgG within immune deposits. Consecutive series of 165 cases of PLA2R-negative MN and 142 MLN biopsies were screened to determine the frequency for each potential antigen. Seven putative antigens were discovered within immune complexes from biopsies of patients with MN including FCN3, CD206, EEA1, SEZ6L2, NPR3, MST1, and VASN. Peptides from these proteins were not enriched in the 278 cases of known antigen type. Between three to 30 unique peptides were detected for each new target. Frequencies of each biomarker, determined by staining consecutive case series, ranged from under 1 to 4.9%. NPR3 and CD206 were only positive in index cases. All cases showed co-localization of IgG within the immune deposits. Thus, seven putative antigens were newly identified in MN and MLN. Due to the number of antigens identified, it is becoming impractical to type PLA2R-negative MN or MLN cases through immunostaining alone. A multiplex approach is needed for subtyping of these diseases. Membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults and carries a high disease burden, with up to 16%–40% of patients developing kidney failure at 5–15 years.1Schieppati A. Mosconi L. Perna A. et al.Prognosis of untreated patients with idiopathic membranous nephropathy.N Engl J Med. 1993; 329: 85-89Crossref PubMed Scopus (371) Google Scholar, 2Lai W.L. Yeh T.H. Chen P.M. et al.Membranous nephropathy: a review on the pathogenesis, diagnosis, and treatment.J Formos Med Assoc. 2015; 114: 102-111Crossref PubMed Scopus (94) Google Scholar, 3Nazareth T.A. Kariburyo F. Kirkemo A. et al.Patients with idiopathic membranous nephropathy: a real-world clinical and economic analysis of U.S. claims data.J Manag Care Spec Pharm. 2019; 25: 1011-1020PubMed Google Scholar It is characterized by the presence of subepithelial immune complex deposits within glomeruli, which disrupt the glomerular filtration barrier and result in proteinuria. Traditionally, this disease has been designated as "primary" when there are no known associated diseases or "secondary" when certain diseases such as systemic lupus erythematosus or malignancy coincide. The main etiologies of "primary MN" were due to autoantibodies directed against the podocyte proteins phospholipase A2 receptor (PLA2R) in nearly 70% of cases4Beck L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1687) Google Scholar and thrombospondin type 1 domain containing 7A (THSD7A) in approximately 3% of total MN.5Tomas N.M. Beck Jr., L.H. Meyer-Schwesinger C. et al.Thrombospondin type-1 domain-containing 7A in idiopathic membranous nephropathy.N Engl J Med. 2014; 371: 2277-2287Crossref PubMed Scopus (635) Google Scholar These renal-limited "primary" forms of MN were identified in 2009 and 2014, respectively. Before these discoveries, only 1 other antigenic type of MN was identified, MN due to antineutral endopeptidase antibodies, a very rare form that manifests in the antenatal and perinatal period.6Debiec H. Guigonis V. Mougenot B. et al.Antenatal membranous glomerulonephritis due to anti-neutral endopeptidase antibodies.N Engl J Med. 2002; 346: 2053-2060Crossref PubMed Scopus (451) Google Scholar Within the past 3 years alone, 10 new target antigens have been described in MN, including exostosin 1/2 (EXT1/2),7Sethi S. Madden B.J. Debiec H. et al.Exostosin 1/exostosin 2-associated membranous nephropathy.J Am Soc Nephrol. 2019; 30: 1123-1136Crossref PubMed Scopus (168) Google Scholar neural epidermal growth factor-like 1 (NELL1),8Sethi S. Debiec H. Madden B. et al.Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy.Kidney Int. 2020; 97: 163-174Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar serine protease HTRA1 (HTRA1),9Al-Rabadi L.F. Caza T. Trivin-Avillach C. et al.Serine protease HTRA1 as a novel target antigen in primary membranous nephropathy.J Am Soc Nephrol. 2021; 32: 1666-1681Crossref PubMed Scopus (50) Google Scholar semaphorin 3B,10Sethi S. Debiec H. Madden B. et al.Semaphorin 3B-associated membranous nephropathy is a distinct type of disease predominantly present in pediatric patients.Kidney Int. 2020; 98: 1253-1264Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar protocadherin 7A (PCDH7A),11Sethi S. Madden B. Debiec H. et al.Protocadherin 7-associated membranous nephropathy.J Am Soc Nephrol. 2021; 32: 1249-1261Crossref PubMed Scopus (64) Google Scholar PCDH FAT1,12Sethi S. Madden B. Casal Moura M. et al.Hematopoietic stem cell transplant—membranous nephropathy is associated with protocadherin FAT1.J Am Soc Nephrol. 2022; 33: 1033-1044Crossref PubMed Scopus (27) Google Scholar netrin G1,13Reinhard L. Machalitza M. Wiech T. et al.Netrin G1 is a novel target antigen in primary membranous nephropathy.J Am Soc Nephrol. 2022; 33: 1823-1831Crossref PubMed Scopus (15) Google Scholar contactin 1 (CNTN1),14Le Quintrec M. Teisseyre M. Bec N. et al.Contactin-1 is a novel target antigen in membranous nephropathy associated with chronic inflammatory demyelinating polyneuropathy.Kidney Int. 2021; 100: 1240-1249Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar neural cell adhesion molecule-1 (NCAM1),15Caza T.N. Hassen S.I. Kuperman M. et al.Neural cell adhesion molecule 1 is a novel autoantigen in membranous lupus nephritis.Kidney Int. 2021; 100: 171-181Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar transforming growth factor beta receptor 3 (TGFBR3),16Caza T.N. Hassen S.I. Kenan D.J. et al.Transforming growth factor beta receptor 3 (TGFBR3)-associated membranous nephropathy.Kidney360. 2021; 2: 1275-1286Crossref PubMed Scopus (23) Google Scholar neural-derived neurotropic factor,17Sethi S. Madden B. Moura M.C. et al.Membranous nephropathy in syphilis is associated with neuron-derived neurotrophic factor. J Am Soc Nephrol.https://doi.org/10.1681/ASN.0000000000000061Google Scholar and proprotein convertase subtilisin/kexin type 6.18Sethi S. Casal Moura M. Madden B. Fervenza F.C. NSAID-associated membranous nephropathy (MN) is associated with PCSK6. American Society of Nephrology Annual Scientific Meeting, 2022Google Scholar The "primary" versus "secondary" classification of MN is becoming obsolete with the discovery of these multiple new antigenic drivers of disease, some of which have unique clinical characteristics or "secondary" associations.19Ronco P. Plaisier E. Debiec H. Advances in membranous nephropathy.J Clin Med. 2021; 10: 607Crossref PubMed Scopus (36) Google Scholar,20Bobart S.A. Tehranian S. Sethi S. et al.A target antigen-based approach to the classification of membranous nephropathy.Mayo Clin Proc. 2021; 96: 577-591Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Despite the growing understanding about the pathogenesis of this disease, the target antigen remains unknown in approximately 15%–20% of cases that were formerly known as "primary" MN and approximately 55% of membranous lupus nephritis (MLN).21Caza T.N. Al-Rabadi L.F. Beck Jr., L.H. How times have changed! A cornucopia of antigens for membranous nephropathy.Front Immunol. 2021; 12800242Crossref PubMed Scopus (8) Google Scholar Knowing the antigenic target of disease can be helpful in monitoring disease activity and may even be useful for primary diagnosis, as has been established for PLA2R-positive MN.22Bech A.P. Hofstra J.M. Brenchley P.E. Wetzels J.F.M. Association of anti-PLA2R antibodies with outcomes after immunosuppressive therapy in idiopathic membranous nephropathy.Clin J Am Soc Nephrol. 2014; 9: 1386-1392Crossref PubMed Scopus (147) Google Scholar, 23Dai H. Zhang H. He Y. Diagnostic accuracy of PLA2R autoantibodies and glomerular staining for the differentiation of idiopathic and secondary membranous nephropathy: an updated meta-analysis.Sci Rep. 2015; 5: 8803Crossref PubMed Scopus (64) Google Scholar, 24Guerry M.J. Vanhille P. Ronco P. Debiec H. Serum anti-PLA2R antibodies may be present before clinical manifestations of membranous nephropathy.Kidney Int. 2016; 89: 1399Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 25Kanigicherla D. Gummadova J. McKenzie E.A. et al.Anti-PLA2R antibodies measured by ELISA predict long-term outcome in a prevalent population of patients with idiopathic membranous nephropathy.Kidney Int. 2013; 83: 940-948Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 26Radice A. Pieruzzi F. Trezzi B. et al.Diagnostic specificity of autoantibodies to M-type phospholipase A2 receptor (PLA2R) in differentiating idiopathic membranous nephropathy (IMN) from secondary forms and other glomerular diseases.J Nephrol. 2018; 31: 271-278Crossref PubMed Scopus (53) Google Scholar In addition, knowledge of the antigenic type often adds information about the etiology of the patient's disease. For example, patients with antibodies directed against CNTN1 often have a chronic inflammatory demyelinating polyneuropathy.14Le Quintrec M. Teisseyre M. Bec N. et al.Contactin-1 is a novel target antigen in membranous nephropathy associated with chronic inflammatory demyelinating polyneuropathy.Kidney Int. 2021; 100: 1240-1249Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Likewise, patients with MN with EXT1/2, NCAM1, or TGFBR3 as a putative antigen often will have a corresponding autoimmune disease, such as systemic lupus erythematosus.7Sethi S. Madden B.J. Debiec H. et al.Exostosin 1/exostosin 2-associated membranous nephropathy.J Am Soc Nephrol. 2019; 30: 1123-1136Crossref PubMed Scopus (168) Google Scholar,15Caza T.N. Hassen S.I. Kuperman M. et al.Neural cell adhesion molecule 1 is a novel autoantigen in membranous lupus nephritis.Kidney Int. 2021; 100: 171-181Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar,16Caza T.N. Hassen S.I. Kenan D.J. et al.Transforming growth factor beta receptor 3 (TGFBR3)-associated membranous nephropathy.Kidney360. 2021; 2: 1275-1286Crossref PubMed Scopus (23) Google Scholar EXT1/2 positivity also carries prognostic value, as patients with EXT1/2 positivity have a reduced risk of progression to end-stage kidney disease.27Ravindran A. Casal Moura M. Fervenza F.C. et al.In patients with membranous lupus nephritis, exostosin-positivity and exostosin-negativity represent two different phenotypes.J Am Soc Nephrol. 2021; 32: 695-706Crossref PubMed Scopus (47) Google Scholar,28Saïdi M. Brochériou I. Estève E. et al.The exostosin immunohistochemical status differentiates lupus membranous nephropathy subsets with different outcomes.Kidney Int Rep. 2021; 6: 1977-1980Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar The identification of other antigenic targets, like NELL1, indicates a higher likelihood of cancer or the use of certain medications, such as lipoic acid, traditional indigenous medicines, or exposure to toxic substances such as mercury.29Spain R.I. Andeen N.K. Gibson P.C. et al.Lipoic acid supplementation associated with neural epidermal growth factor-like 1 (NELL1)-associated membranous nephropathy.Kidney Int. 2021; 100: 1208-1213Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 30Caza T.N. Larsen C.P. Lipoic acid in neural epidermal growth factor-like 1-associated membranous nephropathy: more than a coincidence?.Kidney Int. 2022; 101: 418-419Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 31Pathak N. Gunasekaran I. Ambriose M. Nanda S.K. Nell1 as target antigen for mercury related membranous nephropathy: a case report.Indian J Nephrol. 2022; 32: 502-505Crossref PubMed Scopus (3) Google Scholar, 32Kurien A.A. Jansi Prema K.S. Walker P. Caza T.N. Traditional indigenous medicines are an etiologic consideration for NELL1-positive membranous nephropathy.Kidney Int. 2022; 102: 1424-1426Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar Like many diseases that are diagnosed based on a histopathologic pattern, we now know that MN actually represents many different underlying disease states at the molecular pathophysiological level. The major antigens have now been identified in idiopathic MN, with the remaining being minor antigens in a small subset of patients with the disease. For MLN, most cases are not of known antigen type. All known antigens and putative antigens in MN and MLN were identified by mass spectrometry (MS), the majority of which employed tissue-based approaches. These use residual kidney biopsy tissue to enrich for glomerular proteins by laser capture microdissection and/or through elution of immune complexes through immunoprecipitation (IP) with protein G that binds IgG.21Caza T.N. Al-Rabadi L.F. Beck Jr., L.H. How times have changed! A cornucopia of antigens for membranous nephropathy.Front Immunol. 2021; 12800242Crossref PubMed Scopus (8) Google Scholar Here, we identified multiple novel biomarkers in PLA2R-/THSD7A-/EXT-/NELL1-quadruple negative MN biopsies and MLN biopsies through protein G IP and MS. Each putative antigen was confirmed by confocal microscopy, and the prevalence was screened in cohorts of MN and MLN biopsies. All cases used in this study were obtained from the clinical case archives at Arkana Laboratories. All data were collected according to a study protocol approved by Solutions institutional review board, and all ethical principles and guidelines for the protection of human subjects in research were followed. Kidney biopsies were identified for the MS discovery cohorts from a biopsy database according to the following inclusion criteria: (i) diagnosis of membranous glomerulopathy on kidney biopsy, (ii) ≥10 glomeruli by light microscopy and ≥5 glomeruli by immunofluorescence microscopy, and (iii) ≤50% interstitial fibrosis and tubular atrophy. Cases where MN was not the dominant pathology, including IgG4-related kidney disease, anti–brush border antibody disease, and antineutrophil cytoplasmic autoantibody–associated glomerulonephritis, were excluded from the analysis. We included cases that were PLA2R-positive, THSD7A-positive, EXT1/2-positive, and NELL1-positive as controls for known antigen type. Biopsies negative for all 4 antigens were used for discovery. MS data were collected from 2 cohorts of cases. Discovery cohort 1 consisted of a total of 59 cases including 24 quadruple antigen-negative (PLA2R-/THSD7A-/EXT1/2-/NELL1-negative) MN cases. Discovery cohort 2 consisted of a 364-case cohort that included 118 "quadruple-negative" cases evaluated in parallel to 265 cases of known antigen type. The details of each cohort can be found in Figure 1. Because of differences in MS methods, it is not possible to combine these discovery cohorts. Protein lysates were prepared from residual kidney biopsy tissue frozen in optimized cutting temperature medium. Biopsy cores were washed 4 times at 4 °C with 1 ml of phosphate-buffered saline (PBS) to remove optimized cutting temperature medium, with 5 minutes per wash. After the final PBS wash, the PBS was replaced with 500 μl Pierce IP Lysis Buffer (catalog #87787; Thermo Fisher) with the addition of protease and phosphatase inhibitors (Halt Protease and Phosphatase Inhibitor Cocktail, catalog #78440; Thermo Fisher). One hundred fifty microliters of 0.1 mm glass beads were added to the samples, followed by mechanical disruption of the tissue cores using a Biospec mini-bead beater. Three cycles of "bead beating" were performed, 2 minutes each. After the final "bead beating" step, the samples were centrifuged using a benchtop microcentrifuge to remove large tissue fragments. The supernatant was transferred to low protein binding microfuge tubes containing 50 μl of PBS-washed magnetic protein G Dynabeads (catalog #1003D; Thermo Fisher). IPs were performed by incubation at room temperature for 1 hour with shaking. The samples were exposed to a magnet, with the supernatant removed after incubation. The beads were then washed 4 times with PBS to reduce nonspecific protein binding. After the final wash, the beads were frozen at −20 °C until further analysis. Paraffin-embedded tissue was cut at 10 μm thickness onto Leica PET-membrane frame slides. Twenty to forty glomeruli were dissected per case. These slides were then stained with Meyer's hematoxylin. The glomeruli were microdissected with a Leica DM6000B microscope. Microdissected glomeruli were lysed in 2% sodium dodecylsulfate and 0.1 M dithiothreitol at 99 °C for 1 hour and processed by filter-assisted sample preparation. Clarified lysate was concentrated using Vivacon 500 concentrators with a molecular weight cutoff of 30 kDa (Sartorius). Washes with 8 M urea in 0.1 M Tris/Cl, pH 8.5, were used to remove sodium dodecylsulfate, and the samples alkylated with 0.05 M iodoacetamide. Iodoacetamide was removed by 3 washes with 8M urea/0.1 M Tris/Cl, pH 8.5, followed by 3 washes with 0.05M ammonium bicarbonate. Protein digestion was performed using sequencing-grade trypsin (Promega) at a 40:1 w/w ratio at 37 °C for 16 hours. Peptides were collected by centrifugation and then desalted on C18-Stage tips (Thermo Fisher). Peptides were produced through trypsin digestion of the immune complexes adhered to protein G beads after IP. MS was performed with data-dependent acquisition for the 59-case cohort and with data-independent acquisition (DIA) from the 364-case cohort. Data-dependent acquisition MS methods were previously described.33Caza T.N. Hassen S.I. Dvanajscak Z. et al.NELL1 is a target antigen in malignancy-associated membranous nephropathy.Kidney Int. 2021; 99: 967-976Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar DIA MS was used for analysis as follows. An UltiMate 3000 RSLC nano system (Thermo Scientific) was used to separate tryptic peptides on a 150 × 0.075 mm column packed with a reverse phase XSelect CSH C18 2.5 μm resin. The peptides were eluted with a gradient of buffer A (0.1% formic acid + 0.5% acetonitrile)/buffer B (0.1% formic acid + 99.9% acetonitrile), from a 97:3 ratio to a 60:40 ratio over 1 hour. Electrospray (2.25 kV) was used to ionize the eluted peptides on an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific). Six gas-phase fractions of a pooled sample were acquired on the Orbitrap Exploris mass spectrometer with 4 m/z DIA spectra (30,000 resolution, normalized automatic gain control target 100%, maximum inject time 66 milliseconds) in a staggered window pattern to assemble a chromatogram library.34Searle B.C. Pino L.K. Egertson J.D. et al.Chromatogram libraries improve peptide detection and quantification by data independent acquisition mass spectrometry.Nat Commun. 2018; 9: 5128Crossref PubMed Scopus (239) Google Scholar Precursor spectra were acquired after each DIA duty cycle, spanning the m/z range of the gas-phase fraction (i.e., 496–602 m/z, 596–702 m/z). For wide-window acquisitions, precursor spectra from each DIA cycle were acquired (385–1015 m/z, 60,000 resolution, normalized automatic gain control target 100%, maximum injection time 50 milliseconds), followed by 50 × 12 m/z DIA spectra (12 m/z precursor isolation windows at 15,000 resolution, normalized automatic gain control target 100%, maximum injection time 33 milliseconds) using a staggered window pattern with optimized window placements. ScaffoldDIA (Proteome software) was used to configure DIA library searches. Narrow-window DIA files were searched against the human predicted spectral library from Prosit (2019/04 Uniprot) with 10 ppm precursor and fragment-ion tolerances to generate an empirically corrected chromatogram library with a peptide and protein false discovery rate of 1%. Wide-window DIA files were subsequently searched against the generated empirically corrected chromatogram library. Staining for PLA2R and EXT2 was performed on frozen tissue by immunohistochemistry. Staining for THSD7A, NELL1, EXT1, NCAM1, TGFBR3, vasorin (VASN), ficolin 3 (FCN3), macrophage stimulating 1 (MST1), natriuretic peptide receptor 3 (NPR3), early endosome antigen 1 (EEA1), cluster of differentiation 206 (CD206), and seizure-related 6 homolog like 2 (SEZ6L2) was performed on formalin-fixed paraffin-embedded tissue sections by paraffin immunofluorescence. These primary antibodies were applied after deparaffinization and antigen retrieval. THSD7A, EXT1, NCAM1, TGFBR3, FCN3, CD206, NPR3, SEZ6L2, and MST1 underwent heat-based antigen retrieval at 95 °C using the PT-link system (Leica Biosystems). NELL1, EEA1, and VASN were retrieved with proteinase K. The secondary antibody Alexa Fluor 488 goat anti-mouse IgG was used with the NELL1 antibody. The secondary antibody Rhodamine Red X Affinipure goat anti-rabbit IgG was used for tissue sections exposed to the other primary antibodies (EXT1, NCAM1, TGFBR3, VASN, FCN3, MST1, EEA1, CD206, SEZ6L2, and NPR3). When sufficient frozen tissue was available, staining for IgG subclasses was performed. Frozen tissue sections (4 μM) were washed in PBS to remove optimized cutting temperature medium. Tissue sections were incubated with fluorescein isothiocyanate (FITC)–conjugated IgG1, IgG2, IgG3, and IgG4 antibodies for 30 minutes at room temperature in the dark. Slides were coverslipped in aqueous mounting medium and imaged immediately. All antibodies were commercially available, and the supplier information is as follows: PLA2R rabbit polyclonal antibody (cat #HPA012657, 1:50; Sigma-Aldrich), THSD7A rabbit polyclonal antibody (cat #AMAB91234, 1:50 dilution; Atlas Antibodies), EXT2 rabbit polyclonal antibody (cat #ab102843, 1:50; Abcam), EXT1 rabbit polyclonal antibody (cat #PA5-60699, 1:50 dilution; Invitrogen), NELL1 mouse monoclonal antibody (cat #PA5-27958, 1:50 dilution; Invitrogen), NCAM1 rabbit polyclonal antibody (cat #HPA039835; Sigma-Aldrich), TGFBR3 rabbit polyclonal antibody (1:25 dilution; Thermo Fisher), FCN3 rabbit polyclonal antibody (cat #PA5-71727, 1:50 dilution; Invitrogen), CD206 rabbit polyclonal antibody (cat #PA5-82136, 1:50 dilution; Invitrogen), VASN rabbit polyclonal antibody (cat #PA5-98236, dilution; Invitrogen), MST1/MST1 rabbit polyclonal antibody (cat #PA5-42762, 1:50; Invitrogen), EEA1 rabbit polyclonal antibody (cat #PA5-29013, 1:25 dilution; Invitrogen), NPR3 rabbit polyclonal antibody (cat #PA5-85282, 1:200 dilution; Invitrogen), SEZ6L2 rabbit polyclonal antibody (cat #PA5-64172, 1:50; Invitrogen), Alexa Fluor 488 goat anti-rabbit IgG (cat #111-035-045, 1:100 dilution; Jackson Immunoresearch), FITC-conjugated goat anti-mouse IgG (cat #111-095-003, 1:100 dilution; Jackson Immunoresearch), Rhodamine Red X Affinipure goat anti-rabbit IgG (cat #111-295-144, 1:100 dilution; Jackson Immunoresearch), peroxidase-conjugated goat anti-rabbit IgG (cat #111-035-045, 1:100 dilution; Jackson Immunoresearch), and polyclonal (FITC-conjugated) rabbit anti-human IgG (1:40 dilution; Agilent). IgG subclass antibodies were all direct FITC conjugates from Jackson Immunoresearch (IgG1, catalog #115-095-205; IgG2, catalog #115-095-207; IgG3, catalog #F4641; and IgG4, catalog #F9889, all used at 1:25 dilution). Staining specificity was established by staining 15 known negative control cases for each antigen, including 3 cases each of PLA2R-positive MN, THSD7A-positive MN, EXT1/2-positive MN, NELL1-positive MN, and diabetic nephropathy cases. Granular capillary loop staining was considered a positive result, and cases were considered negative if there was an absence of staining along the glomerular capillary loops. For confocal microscopy, tissue sections were costained with FITC-conjugated polyclonal rabbit anti-human IgG at 1:40 dilution. Slides were coverslipped in antifade mounting medium and imaged under a Leica SBA DMI8 confocal laser scanning microscope. PLA2R-positive MN cases were costained in parallel to cases of interest as negative controls. Retrospective cohorts of consecutive kidney biopsies were evaluated to determine the frequency for each biomarker. These cohorts included 165 PLA2R-negative MN and 142 cases of MLN. Of all idiopathic MN biopsies in the time frame of this consecutive cohort, 29.3% were PLA2R-negative. All cases were stained for each biomarker, including SEZ6L2, VASN, EEA1, MST1, NPR3, FCN3, and CD206. In addition to immunostaining for each of the 7 biomarkers, staining for NELL1, THSD7A, and EXT1 (or EXT2) was performed on the "idiopathic" MN cohort. For the MLN cohort, immunostaining for EXT1 (or EXT2), NCAM1, and TGFBR3 was performed. These controls were used to evaluate for exclusivity or dual positivity with each of the biomarkers. Patient biopsies negative for PLA2R, THSD7A, EXT1/2, and NELL1 ("quadruple-negative" cases, n = 142 total from 2 discovery cohorts) were evaluated by MS to identify new antigenic targets in MN after protein G IP to elute antibodies from frozen biopsy tissue. These were compared with 278 cases of known antigen type, including PLA2R (n = 100), THSD7A (n = 63), EXT1/2 (n = 56), and NELL1 MN (n = 59). These cases were from 2 discovery cohorts due to being within 2 separate MS runs (Figure 1a and b). To consider an enriched protein to be a potential target in MN in our quadruple-negative biopsies, the following 4 criteria were required: (i) identification by MS within protein G immunoprecipitates of frozen biopsy tissue with 3 or more unique peptides, (ii) granular capillary loop staining of the target protein by paraffin immunofluorescence, (iii) confocal microscopy demonstrating IgG colocalization of the target protein along the glomerular capillary loops, and (iv) specificity of staining along glomerular capillary loops compared with PLA2R, THSD7A, EXT1/2, NELL1, and diabetic glomerulopathy controls. For protein candidates that met the above criteria, we screened consecutive case cohorts of PLA2R-negative idiopathic MN and MLN to evaluate the frequency of each putative antigen in disease. To determine whether protein G IP of immune complexes was equivalent to laser capture microdissection (used to enrich glomeruli from tissue samples) in subtyping MN cases by MS, a subset of cases of known antigenic types were examined by both techniques. The same antigen type was identified by protein G IP and laser capture microdissection, demonstrating equal sensitivity. This was observed in 3 cases of PLA2R-positive MN, 3 cases of THSD7A-positive MN, 4 cases of EXT1/2-positive MN, 2 cases of NELL1-positive MN, 3 cases of NCAM1-positive MN, 1 case of HTRA1-positive MN, 2 cases of TGFBR3-positive MN, 1 case of semaphorin 3B–positive MN, 1 case of PCDH7-positive MN, 2 cases of CNTN1-positive MN, and 1 case of netrin G1–positive MN (data not shown). These data were shown previously for PLA2R, THSD7A, EXT1/2, NCAM1, HTRA1, and TGFBR3.9Al-Rabadi L.F. Caza T. Trivin-Avillach C. et al.Serine protease HTRA1 as a novel target antigen in primary membranous nephropathy.J Am Soc Nephrol. 2021; 32: 1666-1681Crossref PubMed Scopus (50) Google Scholar,15Caza T.N. Hassen S.I. Kuperman M. et al.Neural cell adhesion molecule 1 is a novel autoantigen in membranous lupus nephritis.Kidney Int. 2021; 100: 171-181Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar,16Caza T.N. Hassen S.I. Kenan D.J. et al.Transforming growth factor beta receptor